Name: quantitative immunoblotting of cell lines as a standard to validate immunofluorescence for quantifying biomarker proteins in routine tissue samples
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples at JoVE.co

This work was partially funded by the Fredrick Banting and Charles Best Canada Graduate Scholarship (A.M.).

Approval to use primary human tissue samples was obtained from the Health Sciences and Affiliated Teaching Hospitals Research Ethics Board (HSREB) at Queen&#39;s University.
1. Building a Cell-line Tissue Microarray (TMA)
<ol>
	<li>Harvest and wash cells. 
	NOTE: This protocol has been tested on various established immortalized cell lines (e.g. HeLa, Jurkat, RCH-ACV).
	<ol>
		<li>For adherent cells, harvest approximately 1.3 x 107 cells once they reach approximately ...

<ol>
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We have described a method that makes use of quantitative immunoblotting (IB) to demonstrate the utility of immunofluorescence (IF) for ascertaining the relative abundance of a target protein in FFPE tissue samples. Current protein quantification methods are limited by their categorical nature, such as chromogenic IHC2,3, or by the need to homogenize samples, preventing investigation into the sample structure and cell populations, such as with IB and mass spectro...

The need to quantify proteins in formalin-fixed, paraffin-embedded (FFPE) tissue biopsy samples exists in many clinical fields. For example, quantification of biomarker proteins in routine biopsy specimens is used to elucidate prognosis and inform treatment for cancer patients1. However, current methods are typically subjective and lack validation.
Immunohistochemistry (IHC) is used routinely in pathology laboratories and generally depends on a primary antibody directed at the target protein and a secondary antibody conjugated with an enzymatic label such as horseradish peroxidase2. Conventional IHC is sensitive, can make use of minute samples and preserves the morphological integrity of tissue samples thereby permitting assessment of protein expression within its relevant histological context. However, because the chromogenic signal generated by IHC is subtractive, it suffers from a relatively narrow dynamic range and offers limited potential for multiplexing2,3,4. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) preserves morphological integrity. However, this developing technology is associated with modest morphological resolution and requires significant calibration and normalization, impairing its feasibility for routine clinical use5,6,7. Alternative techniques to quantify protein in tissue samples include immunoblotting8, mass spectrometry9,10,11, and enzyme-linked immunosorbent assay (ELISA)12, each of which begins with a homogenized lysate of sample tissue. Primary tissue samples are heterogeneous in that they contain a multitude of cell types. Therefore, techniques that entail homogenizing the samples do not permit quantification of a protein in a particular cell population of interest such as cancer cells.
Like IHC, IF is applicable to small FFPE samples and permits the retention of histological integrity13. However, thanks to the additive nature of fluorescence signals, IF is amenable to the application of multiple primary antibodies and fluorescent labels. Thus, a protein of interest may be relatively quantified within specific cells or cellular compartments (for example, nucleus versus cytoplasm) defined using other antibodies. Fluorescence signals also have the advantage of a greater dynamic range13,14. The superiority, reproducibility, and multiplexing potential of IF applied to FFPE samples has been demonstrated13,14,15.
Herein we describe the use of quantitative immunoblotting using established cell lines as a gold standard to ascertain the quantitative nature of IF coupled with computer-assisted image analysis in determining the relative abundance of a protein of interest in histological sections from FFPE tissue samples. We have applied this method successfully in a multiplex approach to quantify biomarker proteins in clinical biopsy samples16,17,18.

<table border="0" cellpadding="0" cellspacing="0" style="width:675px;" width="673">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">697</td>
			<td style="width:116px;">DSMZ</td>
			<td style="width:123px;">ACC 42</td>
			<td style="width:213px;">Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">JeKo-1</td>
			<td style="width:116px;">ATCC</td>
			<td style="width:123px;">CRL-3006</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Jurkat</td>
			<td style="width:116px;">ATCC</td>
			<td style="width:123px;">TIB-152</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">RCH-ACV</td>
			<td style="width:116px;">DSMZ</td>
			<td style="width:123px;">ACC 548</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Granta-519</td>
			<td style="width:116px;">DSMZ</td>
			<td style="width:123px;">ACC 342</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">REH</td>
			<td style="width:116px;">DSMZ</td>
			<td style="width:123px;">ACC 22</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Raji</td>
			<td style="width:116px;">ATCC</td>
			<td style="width:123px;">CCL-86</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">HeLa</td>
			<td style="width:116px;">ATCC</td>
			<td style="width:123px;">CCL-2</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Trypsin/EDTA solution</td>
			<td style="width:116px;">Invitrogen</td>
			<td style="width:123px;">R001100</td>
			<td>For detaching adhesive cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Fetal bovine serum (FBS)</td>
			<td style="width:116px;">Wisent Inc.</td>
			<td style="width:123px;">81150</td>
			<td>To neutralize trypsin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Neutral Buffered Formalin</td>
			<td style="width:116px;">Protocol</td>
			<td style="width:123px;">245-684</td>
			<td>For fixing cell pellets</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">UltraPure low melting point agarose</td>
			<td style="width:116px;">Invitrogen</td>
			<td style="width:123px;">15517-022</td>
			<td>For casting cell pellets</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">Mouse monoclonal anti-human Bcl-2 antibody, clone 124</td>
			<td style="width:116px;">Dako (Agilent)</td>
			<td style="width:123px;">cat#: M088729-2, RRID: 2064429</td>
			<td style="width:213px;">To detect Bcl-2 by immunoflourencence and immunoblot (lot#:&#160;00095786</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Ventana Discovery XT</td>
			<td style="width:116px;">Roche</td>
			<td style="width:123px;">-</td>
			<td style="width:213px;">For automation of immunofluorescence staining</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">EnVision+ System- HRP labelled polymer (anti-mouse)</td>
			<td style="width:116px;">Dako (Agilent)</td>
			<td style="width:123px;">K4000</td>
			<td style="width:213px;">For immunofluorescence signal amplification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">EnVision+ System- HRP labelled polymer (anti-rabbit)</td>
			<td style="width:116px;">Dako (Agilent)</td>
			<td style="width:123px;">K4002</td>
			<td style="width:213px;">For immunofluorescence signal amplification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Cyanine 5 tyramide reagent</td>
			<td style="width:116px;">Perkin Elmer</td>
			<td style="width:123px;">NEL745001KT</td>
			<td style="width:213px;">For immunofluorescence signal amplification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Aperio ImageScope</td>
			<td style="width:116px;">Leica Biosystems</td>
			<td style="width:123px;">-</td>
			<td style="width:213px;">To view scanned slides</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">HALO image analysis software</td>
			<td style="width:116px;">Indica Labs</td>
			<td style="width:123px;">-</td>
			<td style="width:213px;">For quantification of immunofluorescence</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">Protease inhibitors (Halt protease inhibitor cocktail, 100X)</td>
			<td style="width:116px;">Thermo Scientific</td>
			<td style="width:123px;">1862209</td>
			<td>To add to RIPA buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:116px;">BioShop</td>
			<td style="width:123px;">EDT001</td>
			<td>For RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">NP-40</td>
			<td style="width:116px;">BDH Limited</td>
			<td style="width:123px;">56009</td>
			<td>For RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Sodium deoxycholate</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:123px;">D6750</td>
			<td>For RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Glycerol</td>
			<td style="width:116px;">FisherBiotech</td>
			<td style="width:123px;">BP229</td>
			<td>For Laemlli buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Bromophenol blue</td>
			<td style="width:116px;">BioShop</td>
			<td style="width:123px;">BRO777</td>
			<td>For Laemlli buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Dithiothreitol (DTT)</td>
			<td style="width:116px;">Bio-Rad</td>
			<td style="width:123px;">161-0611</td>
			<td>For Laemlli buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Bovine serum albumin (BSA)</td>
			<td style="width:116px;">BioShop</td>
			<td style="width:123px;">ALB001</td>
			<td>For immunoblot washes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Protein ladder (Precision Plus Protein Dual Color Standards)</td>
			<td style="width:116px;">Bio-Rad</td>
			<td style="width:123px;">161-0374</td>
			<td>For running protein gel</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Filter paper (Extra thick blot paper)</td>
			<td style="width:116px;">Bio-Rad</td>
			<td style="width:123px;">1703969</td>
			<td>For blotting transfer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Nitrocellulose membrane</td>
			<td style="width:116px;">Bio-Rad</td>
			<td style="width:123px;">162-0115</td>
			<td>For blotting transfer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Trans-blot SD semi-dry transfer cell</td>
			<td style="width:116px;">Bio-Rad</td>
			<td>1703940</td>
			<td>For semi-dry transfer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Skim milk powder (Nonfat dry milk)</td>
			<td style="width:116px;">Cell Signaling Technology</td>
			<td style="width:123px;">9999S</td>
			<td>For blocking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Tween 20</td>
			<td style="width:116px;">BioShop</td>
			<td style="width:123px;">TWN510</td>
			<td>For wash buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">GAPDH rabbit monoclonal antibody</td>
			<td style="width:116px;">Epitomics</td>
			<td style="width:123px;">2251-1</td>
			<td style="width:213px;">Primary antibody of control protein (lot#: YE101901C)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Goat anti-mouse IgG (HRP conjugated) antibody</td>
			<td style="width:116px;">abcam</td>
			<td style="width:123px;">cat#: ab6789, RRID: AB_955439</td>
			<td style="width:213px;">Secondary antibody for immunoblot</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">Goat anti-rabbit IgG (HRP conjugated) antibody</td>
			<td style="width:116px;">abcam</td>
			<td style="width:123px;">cat#: ab6721, RRID: AB_955447</td>
			<td style="width:213px;">Secondary antibody for immunoblot (lot#: GR3192725-5)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Clarity Western ECL substrates</td>
			<td style="width:116px;">Bio-Rad</td>
			<td style="width:123px;">1705060</td>
			<td style="width:213px;">For immunoblot signal detection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Amersham Imager 600</td>
			<td style="width:116px;">GE Healthcare Life Sciences</td>
			<td style="width:123px;">29083461</td>
			<td>For immunoblot signal detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">ImageJ software</td>
			<td style="width:116px;">Freeware, NIH</td>
			<td style="width:123px;">-</td>
			<td>For densitometry analysis</td>
		</tr>
	</tbody>
</table>

quantitative immunoblotting of cell lines as a standard to validate immunofluorescence for quantifying biomarker proteins in routine tissue samples

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An optimal method of quantification is accurate, has a broad linear dynamic range and maintains the structural integrity of the sample to allow for identification of individual cell types. Current methods such as immunohistochemistry (IHC), mass spectrometry, and immunoblotting each fail to meet these stipulations due to their categorical nature or need to homogenize the sample. As an alternative method, we propose the use of immunofluorescence (IF) and image analysis to determine the relative abundance of a protein of interest in FFPE tissues. Herein we demonstrate that this method is easily optimized, yields a wide dynamic range, and is linearly quantifiable as compared to the gold standard of quantitative immunoblotting. Furthermore, this method permits the maintenance of the structural integrity of the sample and allows for the distinction of various cell types, which may be crucial in diagnostic applications. Overall, this is a robust method for the relative quantification of proteins in FFPE samples and can be easily adapted to suit clinical or research needs.

This protocol was used to confirm the ability of IF to determine the relative quantity of the anti-apoptotic protein Bcl-2 in cell lines made into FFPE tissue blocks. Quantifying Bcl-2 selectively in cancer cells can elucidate oncogenic mechanisms and can be useful in pathological diagnosis and in informing clinical management decisions24. More specifically, Bcl-2 plays a role in proper B-lymphocyte development and its expression is commonly investigated in the con...

Watch this Scientific Journal Video about Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples at JoVE.co

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples

We describe the use of quantitative immunoblotting to validate immunofluorescence histology coupled with image analysis as a means of quantifying a protein of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Our results demonstrate the utility of immunofluorescence histology for ascertaining the relative quantity of biomarker proteins in routine biopsy samples.

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An ...

Research

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Biology

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...